Adenoviruses (Ads) are a family of DNA viruses characterized by icosahedral, non-enveloped capsids containing a linear DNA genome.
The human adenovirus type 5 (Ad5) has a linear, double-stranded genome of approximately 36 kb, divided into early and late viral functions (see Berkner 1992, Curr. Topics Micro. Immunol. 158:39-66). A representative Adenovirus 5 ("Ad5") genome for use with the embodiments of the present invention is a 36 kB linear duplex. Its sequence has been published. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology186, 280-285; hereby incorporated by reference).
Upon infection of permissive cells, the first region transcribed from the Ad5 viral genome, E1A at the left end of the conventional map, encodes proteins that are involved in transactivation of other viral early and late genes. E1B, also at the left end of the genome, encodes proteins that regulate host cell and viral RNA and protein synthesis, and protect cells from E1A-induced apoptosis. Thus, E1 functions encoded by E1A and E1B are essential for viral replication. E1-deleted virus can be propagated in the 293 cell line which contains and expresses E1 of Ad5 (Graham et al. 1977).
Removal of the essential early regions 1A and 1B (E1A and E1B) of Ad5 generates conditional helper-independent Ads that can be grown and propagated in the E1-complementing 293 cell line (Graham et al. 1977, J. Gen. Virol, 36:59-72). Foreign genes have been cloned into the replication-defective Ads, and these vectors have been used extensively for the delivery of genes into mammalian cells for gene therapy, as recombinant viral vaccines, or for general purpose expression vectors for experimental studies. Ads also have the advantage that they are well characterized both genetically and biochemically, easy to manipulate, and can be grown to a very high titer. Furthermore, adenovirus is a relatively safe vector that has not been associated with any neoplastic disease, and usually causes relatively mild infections in immuno-competent individuals.
E1-deleted Ad vectors can accommodate DNA inserts of .about.4.7 kb (up to 105% of the wild-type genome), and deletions in the non-essential E3 region can further increase the cloning capacity to .about.8 kb (Bett et al. 1993, J. Virol. 67:5911-5921). However, Ad vectors with DNA inserts that increase the genome size to greater than 105% of wild-type DNA content are either non-viable or unstable, and frequently undergo DNA rearrangements to reduce the overall size of the vector (Ghosh-Choudhury et al. 1987, EMBO J. 6:1733-1739; Bett et al. 1993, J. Virol. 67:5911-5921). This is presumably due to a destabilization of the capsid because of the increased DNA content. Thus, the size of DNA inserts in "first generation" Ad vectors (i.e., E1-deleted and/or E3 deleted) is limited by the necessity to retain sufficient Ad coding sequences to allow helper-independent growth, limiting the size of "non-essential" regions that can be deleted from the genome, and the need to maintain virion stability.
Stability of the adenovirus capsid is conferred, at least in part, by protein IX (pIX). pIX has been shown to be associated with the hexons that make up the "facets" of the icosahedron (Furcinitti et al. 1989, EMBO J. 8:3563-3570) Although originally thought to be dispensable for virion formation (Colby and Shenk 1981, J. Virol. 39:977-980), pIX is required for the packaging of full-length viral DNA molecules (Haj-Ahmad and Graham 1986, J. Virol. 57:267-274). Deletion or inactivation of pIX results in virions that are heat labile with capsids that can accommodate only 35 kb of viral DNA (.about.97% of the wild-type genome). Thus, deletion or inactivation of pIX provides a means of selecting for virions that contain viral DNA that is less than the size of the wild-type genome.
Previously, the lower limit of adenovirus DNA necessary to achieve Ad DNA packaging could not be identified due to the necessity for retaining sufficient protein-coding regions to enable the production of all of the proteins required for Ad DNA replication and virion formation. The development of helper-dependent systems has alleviated this problem. In the helper-dependent systems, a helper virus provides all of the functions necessary in trans for the packaging of an helper-dependent vector, which lacks virtually all virus specific coding sequences. The helper-dependent vector contains only those cis-acting elements required for viral DNA replication and packaging. Since the sequences required for Ad DNA replication and packaging are contained within .about.500 bp of the left and right ends of the genome (Grable and Hearing 1992, J. Virol. 66:723-731), helper-dependent vectors can, in theory, range in size from a few hundred base pairs to greater than the size of wild-type Ad, potentially carrying up to .about.37 kb of foreign DNA. However, it has been demonstrated that Ad vectors that have substantially less DNA than wild-type Ads undergo DNA rearrangements and multimerization (Fisher et al. 1996, Virol. 217:11-22).
Despite all of the advantages of first generation Ad vectors as vectors for the delivery of foreign genes into mammalian cells, current helper-independent vectors retain many viral genes that, when expressed at low levels, may contribute to the induction in the host of an immune response against the transduced cell (Dong et al. 1996, Hum. Gene Ther. 7:319-33 1), resulting in the elimination of the transduced cell. The immune response will ultimately limit the usefulness of current vectors for the treatment of genetic diseases, such as cystic fibrosis, due to the requirement for long term, stable expression in order to correct the genetic deficiencies. Attempts to reduce the expression of viral genes, by the elimination of most, if not all, viral-specific coding sequences, have led to the development of the helper-dependent systems for the generation of Ad vectors (Mitani et al. 1995, Proc. Natl. Acad. Sci. 92:3854-3858; Fisher et al. 1996, Virol. 217:11-22; Kochanek et al. 1996, PNAS 92:5731-5736; Parks et al. 1996, Proc. Natl. Acad. Sci. in press). Previously, we developed a helper-dependent system that utilized a helper virus that had a packaging signal flanked by loxP sites (Parks et al. 1996, Proc. Natl. Acad. Sci. in press). The general principle is outlined in FIG. 1. Upon infection of a 293 cell line that constitutively expressed the Cre recombinase (293Cre; Chen and Graham, unpublished results), the packaging signal was efficiently excised from the helper virus rendering it unpackageable. However, the helper virus DNA was able to replicate and provide all of the functions necessary in trans for the packaging of a helper-dependent vector, which contained only those cis-acting elements required for viral DNA replication and packaging. Serial passage of the helper-dependent vector in helper-virus infected 293Cre cells allowed us to produce large quantities of the helper-dependent vector (10.sup.10 transducing particles from 4.times.10.sup.8 293Cre cells with an initial level of contamination with helper virus of approximately 0.3-1). After fractionation on CsCl buoyant density gradients, final vector preparations contained less than 0.01 helper virus contamination, a level that is lower than in all other helper-dependent systems reported to date. The contamination of vector with helper virus that is observed is caused by helper virus DNA (.about.10%) that escapes the Cre-mediated excision event, and can therefore be packaged into infectious virions. At present, it is not known why these DNAs are not cleaved by Cre, but it may be due to saturation of the Cre protein in the 293Cre cells. Regardless of the reason for the helper virus contamination of vector stocks, it is apparent that modifications to the system are desired to eliminate the remaining helper virus. It is an object of the present invention to provide an improved method for preparing helper-dependent vectors. The invention herein may be used independently for vector growth or may be combined with the Cre/loxP helper-dependent system to provide a means for vector production without contaminating helper virus.